Abstract

Francisella tularensis is the causative agent of zoonotic tularemia, a severe pneumonia in humans, and Francisella novicida causes a similarly severe tularemia in mice upon inhalation. The correlates of protective immunity, as well as the virulence mechanisms of this deadly pathogen, are not well understood. In the present study, we compared the host immune responses of lethally infected and vaccinated mice to highlight the host determinants of protection from this disease. Intranasal infection with an attenuated mutant (Mut) of F. novicida lacking a 58-kDa hypothetical protein protected C57BL/6 mice from a subsequent challenge with the fully virulent wild-type strain U112 via the same route. The protection conferred by Mut vaccination was associated with reduced bacterial burdens in systemic organs, as well as the absence of bacteremia. Also, there was reduced lung pathology and associated cell death in the lungs of vaccinated mice. Both vaccinated and nonvaccinated mice displayed an initial 2-day delay in upregulation of signature inflammatory mediators after challenge. Whereas the nonvaccinated mice developed severe sepsis characterized by hypercytokinemia and T-cell depletion, the vaccinated mice displayed moderated cytokine induction and contained increased numbers of αβ T cells. The recall response in vaccinated mice consisted of a characteristic Th1-type response in terms of cytokines, as well as antibody isotypes. Our results show that a regulated Th1 type of cell-mediated and humoral immunity in the absence of severe sepsis is associated with protection from respiratory tularemia, whereas a deregulated host response leading to severe sepsis contributes to mortality.

The causative agent of respiratory tularemia, Francisella tularensis, is a gram-negative intracellular bacterium. There are four closely related subspecies of F. tularensis, F. tularensis subsp. tularensis (type A), F. tularensis subsp. holarctica (type B), F. tularensis subsp. mediasiatica, and “F. tularensis subsp. novicida,” and type A is the most virulent subspecies in humans (20). This pathogen is capable of causing acute respiratory infection following inhalation of as few as 10 organisms (10, 48). This extremely low infectious dose, the ease of transmission via the aerosol route, and the wide host range have led the CDC to recognize this pathogen as a potential bioweapon (56). Since the fully virulent strains of F. tularensis are highly infectious, much of our knowledge about Francisella pathogenesis has been obtained by using the attenuated live vaccine strain (LVS) derived from a type B strain of F. tularensis or Francisella novicida. Although attenuated for humans, F. novicida is virulent in mice and results in a disease that closely resembles human tularemia. Despite continuous efforts, an effective vaccine for tularemia has not been developed yet. This highlights the need for understanding the virulence mechanisms of Francisella, as well as the correlates of protective immunity, in order to devise effective therapeutics for use against tularemia.

Primary respiratory infections with Francisella cause a delay in the initial innate immune response. This initial delay has been postulated to be an important virulence mechanism of the organism (2, 3, 39, 40). An absence of this initial immune response is thought to aid rapid multiplication of bacteria, followed by dissemination of the bacteria to systemic organs, resulting in bacteremia. This causes widespread upregulation of multiple cytokines and chemokines that reflects contributions from both the host and the pathogen to an inappropriate inflammatory response (40, 59, 64). This kind of unbridled host response to a pathogen is now broadly accepted as the cause of host death in infectious diseases like malaria, influenza, and sepsis (6). In light of the absence of any known endo- or exotoxin activity of any virulence factor of Francisella, this hyperimmune response seems to be the cause of the mortality associated with respiratory tularemia (54).

Adaptive immune responses following vaccination, as well as during sublethal infections, have highlighted the contributions of both B and T lymphocytes (8, 16, 44, 53). Most of the studies have been carried out with type B-infected humans, as well as mice (65). Both humans and mice develop antigen-specific antibodies, as well as CD4+ and CD8+ T cells, during sublethal infections (15, 17, 26, 57). The effector T-cell mechanisms that control the infections involve mainly gamma interferon (IFN-γ) and/or tumor necrosis factor alpha (TNF-α) (9, 66), but bacterial killing is partially mediated by NO produced by IFN-γ-activated macrophages (4, 14). However, a comprehensive study of the mechanisms triggering rapid death following systemic dissemination of bacteria before the onset of acquired immunity and the factors involved in bacterial clearance and host protection from lethal respiratory infection in the same experimental setting has not been done.

Analysis of the genome sequence of Francisella revealed a family of five hypothetical proteins unique to this organism (38). One of these factors, a protein encoded by the FTT_0918 gene, has been shown to be a virulence factor, as mutants of type A strains lacking this gene are attenuated for infection in vitro and in vivo. In addition, intradermal inoculation with this mutant protects mice from intranasal challenge with virulent type A strains (63, 65). Our in vivo studies with the murine model organism F. novicida have shown that a transposon mutant (Mut) lacking a homolog of this 58-kDa protein is equally attenuated (54). In the current study we tested this mutant to determine whether it protects against murine respiratory tularemia and determined the host immune responses associated with protection. Intranasal immunization of C57BL/6 mice with Mut protected the mice from a subsequent challenge with an otherwise lethal dose of the wild-type (WT) bacteria. Importantly, the severe sepsis characterized by hypercytokinemia and bacteremia observed in nonvaccinated mice was not present in lungs of mice vaccinated with the mutant. Instead, a protective Th1 type of cytokine and antibody response was upregulated. Our results show that in the apparent absence of any endotoxins or exotoxins that could account for the lethality associated with respiratory tularensis, severe sepsis coupled with a lack of adaptive responses due to T-cell depletion is likely the major contributor to the severity of the disease and associated mortality, and an effective Th1 type of response coupled with the absence of severe sepsis and bacteremia is key for protection against this deadly infection.

MATERIALS AND METHODS

Bacterial strains and mice.

F. novicida WT strain U112 and a transposon mutant lacking the 58-kDa protein mentioned above (locus tag FTN_0444) were kindly provided by L. Gallagher, University of Washington (22). The mutant lacking the 58-kDa protein was provided as a two-allele set with transposon insertions at positions 349 and 962 in the 1,671-nucleotide open reading frame. The insertion positions were confirmed by PCR amplification as described in the F. novicida transposon mutant collection resource (http://francisella.org/transposons.htm). The bacteria were grown on Trypticase soy agar at 37°C. After overnight growth, the bacteria were harvested and suspended in a freezing medium (250 mM sucrose, 10 mM sodium phosphate [pH 7.2], 5 mM glutamic acid). Stock preparations were aliquoted and frozen at −80°C until further use.

All the in vivo experiments were performed using 6- to 8-week-old female C57BL/6 mice purchased from Charles River Laboratories, Wilmington, MA. The animal usage protocols were approved by the Institutional Animal Care and Usage Committee at University of Texas at San Antonio and followed federal guidelines.

Infection of mice.

Mice were anesthetized with a mixture of ketamine HCl and xylazine (30 mg/ml ketamine and 4 mg/ml xylazine in phosphate-buffered saline [PBS]) and were infected intranasally with 3 × 102 CFU of mutant bacteria in 20 μl of PBS or inoculated with 20 μl of PBS alone. Three weeks postinfection (p.i.) the mice were challenged with 3 × 102 CFU of WT bacteria. In our hands, this dose of WT bacteria in unvaccinated mice resulted in 100% mortality by 96 to 120 h p.i. The mock-infected mice received 20 μl of PBS intranasally.

Protection and bacterial burden.

Mice were monitored daily for signs of disease, which typically included piloerection, a hunched gait, lethargy, and eye discharge. The survival of vaccinated mice was recorded for up to 4 weeks postchallenge (p.c.). In some experiments, the mice were euthanized at 6, 24, 72, and 120 h p.c., and blood was collected before the mice were perfused with sterile PBS. The lungs were harvested after perfusion and homogenized aseptically in cold PBS with Complete protease inhibitor cocktail (Roche Diagnostics, Germany). For the bacterial burden analyses, the homogenates were serially diluted in PBS and plated on Trypticase soy agar. The numbers of CFU per mouse were calculated after the plates were incubated at 37°C overnight. The blood collected from animals was plated similarly to determine the numbers of CFU.

FACS analysis.

Lungs were harvested from mice at 72 h p.i. after perfusion with PBS and treated with collagenase to obtain lung cells as previously described (24). To prepare samples for FACS, cells were first treated with Fc receptor blocking antibodies for 15 min at 4°C, which was followed by staining with fluorescently conjugated antibodies at 4°C for 15 min. FACS analysis was performed using a FACSArray (Becton Dickinson) FACS analysis machine equipped with 532- and 635-nm lasers. Cells were stained for lymphoid or myeloid cell-specific markers. The lymphoid cell-specific marker analyzed was αβ TCR, CD4 and CD8, and the myeloid cell-specific markers analyzed were CD11b and GR-1. Appropriate isotype control antibodies were used to determine the levels of background staining. The FlowJo (Tree Star) software was used to analyze the FACS data.

Multianalyte profile analysis.

The lung homogenates were centrifuged at 2,000 × g for 15 min to clear the cellular debris, and the supernatants were immediately frozen at −80°C. The biomarker levels in lung homogenates were determined commercially by Rules-Based Medicine (Austin, TX), utilizing a multiplexed flow-based system, the Mouse MAP (multianalyte Profile) analysis technology.

Enzyme-linked immunosorbent assay (ELISA).

A total of 5 × 106 CFU of WT bacteria was coated onto Maxisorp 96-well plates (BD Biosciences) in 100 μl of carbonate buffer (0.05 M NaHCO3, 0.05 M Na2CO3; pH 9.4) for 2 h at 37°C. The plates were washed with PBS containing 0.1% Tween 20 and blocked with 5% bovine serum albumin in PBS. Twofold serial dilutions of sera were loaded in the plates and incubated for 2 h at 37°C, which was followed by washing and addition of HRP-conjugated goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA antibodies (SouthernBiotech, Birmingham, AL). The plates were incubated for 1 h at 37°C and washed, and then a 3,3′,5,5′-tetramethyl benzidine peroxidase substrate solution was added. After color development, the reaction was stopped with 1.8 N H2SO4, and the optical density at 450 nm was determined. The titers were expressed as the lowest dilutions of serum that gave an optical density that was 0.2 U greater than that of the normal serum control.

Immunoblotting.

Equal amounts of lung homogenates were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk in PBS for 1 h and then incubated with the appropriate dilution of goat anti-S100A9 antibody in 1% skim milk in PBS plus 0.05% Tween 20. The membranes were then incubated with donkey anti-goat IgG conjugated to HRP for 1 h. The signal was detected by enhanced chemiluminescence (Amersham Biosciences). The same blots were probed with anti-glyceraldehyde-3-phosphate dehydrogenase antibody to confirm that there was equal loading of samples in all lanes.

Statistical analysis.

Statistical comparisons of data for different experimental groups were performed with Student's t test using Sigma Plot 8.0.

RESULTS

C57BL/6 mice that were either immunized intranasally with 3 × 102 CFU of the attenuated mutant or treated intranasally with PBS alone were challenged 3 weeks later via the same route with 3 × 102 CFU (100% lethal dose) of fully virulent WT bacteria. Sham-inoculated mice that were challenged intranasally with 3 × 102 CFU of WT bacteria (PBS/WT mice) developed respiratory tularemia and overt signs of the disease by 72 h p.c. that were characterized by piloerection and lethargy. The mice succumbed to infection by 120 h p.c. (Fig. ​(Fig.1A).1A). In contrast, the vaccinated mice (Mut/WT mice) displayed no signs of the disease after WT challenge and appeared to be healthy throughout the period that they were monitored (Fig. ​(Fig.1A).1A). The vaccinated mice survived challenge doses up to 4.8 × 103 CFU of WT bacteria (Fig. ​(Fig.1A)1A) without displaying any signs of disease. In PBS/WT mice, substantial growth of bacteria was detected in the lungs at 72 h p.c. (1.0 × 109 ± 7.4 × 108 CFU), and the bacterial burden remained high at 120 h p.c. (1.6 × 109 ± 1.6 × 1010 CFU), a time when the mice were either moribund or had succumbed to the infection (Fig. ​(Fig.1B).1B). In contrast, vaccination with the mutant considerably reduced the bacterial loads in the lungs of the challenged mice (Mut/WT mice). The bacterial burdens in the lungs of Mut/WT mice were 2 to 3 logs less than those in the lungs of the PBS/WT mice at 6 h, 24 h, and 72 h p.c. (Fig. ​(Fig.1B).1B). Importantly, the bacterial load in the lungs of Mut/WT mice started to decrease by 120 h p.c., suggesting that there was resolution of the infection.

Intranasal vaccination with the mutant lacking the 58-kDa protein gene protects mice from a lethal challenge with F. novicida. Mice were inoculated intranasally with 3 × 102 CFU of Mut bacteria in 20 μl of PBS or with PBS alone. Three...

A hallmark of F. novicida infection in mice is a late-stage bacteremia where the infection becomes systemic and substantial numbers of bacteria are detected in the blood. We could not recover any bacteria in the blood from Mut/WT mice at any time after infection tested, suggesting that the infection did not result in bacteremia. On the other hand, 1.2 × 104 ± 8.5 × 103 CFU/ml blood of PBS/WT mice were recovered at 72 h p.c., and the burden increased to 6.7 × 104 ± 5.5 × 104 CFU/ml of blood at 120 h p.c. (Fig. ​(Fig.1C1C).

Survival of vaccinated mice correlated with reduced lung pathology.

Histopathological examination of lungs from PBS/WT mice showed that there was a delayed response that was characterized by an absence of cellular infiltration (Fig. 2B1 and B2), similar to the response of mock-infected control animals (Fig. 2A1 and A2), until 72 h p.c. This delayed response was consistent with our previous observations (40, 54). Significant cellular infiltration and extensive pathology were evident in the lungs of PBS/WT mice at 72 h p.c., and severe bronchopneumonia and massive cell death occurred in the center of large granuloma-like areas of infiltration (Fig. 2B3). Lungs from Mut/WT mice were clear of cellular infiltrates at 6 h p.c. (Fig. 2C1). However, at 24 h p.c. occasional peribronchial cellular aggregates, mainly neutrophils, were observed (Fig. 2C2). At 72 h p.c. large areas of peribronchial and perivascular infiltration were observed in Mut/WT mice (Fig. 2C3). The infiltrating cells in these areas appeared to be viable, and the areas of infiltration were clear of cellular debris typical of extensive apoptosis and necrosis. To further analyze the extent of cell death, the lungs of PBS/WT and Mut/WT mice were analyzed by using the TUNEL assay at 72 h p.c. As shown in low-magnification images in Fig. ​Fig.3,3, the number of apoptotic TUNEL-positive cells in Mut/WT mice was much less than the number of apoptotic TUNEL-positive cells in the PBS/WT mice. Consistent results were obtained for several mice. The reduced cell death in Mut/WT mice may account for the much better lung architecture during WT challenge.

Mice vaccinated with the mutant exhibit reduced cell death after challenge with the WT bacteria. Lungs from mice vaccinated with the Mut (Mut/WT) or PBS (PBS/WT) were harvested 72 h after challenge with the WT bacteria, embedded in optimal-cutting-temperature...

Altered kinetics of cellular infiltrates after vaccination with the mutant.

An in situ IF analysis was performed with frozen lung tissue sections to examine the kinetics and distribution of granulocytes (Gr1+), monocytes (Cd11b+), and αβ T cells, three types of cells shown to play a role in Francisella pathogenesis (16). To quantify these cells, FACS analysis was used (see below). The infiltration of Gr1+ granulocytes in lung sections of PBS/WT mice (Fig. 4A2 and A3) was similar to that in mock-infected control mice (Fig. 4A1 and A1′) at 6 h and 24 h p.c., whereas the number of these cells at 72 h p.c. was substantially greater in PBS/WT mice than in control mice (Fig. 4A4). These cells were localized mostly in peribronchial lesion areas in PBS/WT mice. On the other hand, the number of Gr1+ cells in the lungs of Mut/WT mice was similar to the number of these cells in the lungs of mock-infected control animals at 6 h p.c. (Fig. 4A2′); however, infiltration of these cells could be observed at 24 h p.c. (Fig. 4A3′). The infiltrating cells were observed mostly in perivascular areas at this time point. At 72 h p.c. the number of Gr1+ cells was increased further in Mut/WT mice, and these cells were present in both perivascular and peribronchial regions of the lungs (Fig. 4A4′). A close look at these cells using a higher magnification showed that they appeared to be viable based on nuclear morphology (Fig. 4A4′, inset). This finding was in contrast to the finding for cells in PBS/WT mice, where the Gr1+ staining appeared to be amorphous in the center of the lesions due to extensive cell death (Fig. 4A4, inset). Similar findings were obtained with CD11b+ cells, and substantial infiltration of these cells was found only at 72 h p c. in both PBS/WT and Mut/WT mouse lungs (data not shown).

Infiltration of Gr1+ cells in lungs of mock-infected, Mut/WT, and PBS/WT mice. Mice were vaccinated intranasally with 3 × 102 CFU of the Mut bacteria in 20 μl of PBS or with 20 μl of PBS alone. Three weeks later mice were...

A similar delay in infiltration of the αβ T cells was observed in lungs from both PBS/WT mice (Fig. 5A2 and A3) and Mut/WT mice (Fig. 5A2′ and A3′), and the numbers of these cells at 6 h and 24 h p.c. did not differ much from the numbers in mock-infected control mice (Fig. 5A1 and A1′). However, at 72 h p.c., the number of αβ T cells was significantly higher in Mut/WT mice (Fig. 5A4′) than that in the mock-infected control mice or PBS/WT mice (Fig. 5A4). The number of αβ T cells in PBS/WT mouse lungs at 72 h p.c. was even less than that in the lungs of mock-infected control mice. This result is consistent with our previous studies in which lethal respiratory infection with F. novicida was shown to deplete αβ T cells from lungs due to extensive apoptosis (55). In the present study, intranasal vaccination with Mut resulted in a substantial increase in the number of αβ T cells in the lungs, indicating that these cells have a role in protection from lethal F. novicida infection.

Infiltration of αβ T cells in lungs of mock-infected, Mut/WT, and PBS/WT mice. Lungs from Mut- or PBS-inoculated mice were harvested at 72 h after challenge with the WT bacteria. Mock-infected control mice received 20 μl of PBS...

FACS analysis of lung cells from PBS/WT and Mut/WT mice confirmed the observations made using IF staining (Fig. ​(Fig.6).6). The PBS/WT mouse lungs displayed significantly greater infiltration of Gr1+ cells (Fig. ​(Fig.6,6, upper panel) and CD11b+ cells (Fig. ​(Fig.6,6, middle panel) than the lungs from mock-infected and Mut/WT mice and exhibited depletion of αβ T cells (Fig. ​(Fig.6,6, lower panel). The Mut/WT mice, on the other hand, had a significantly higher number of αβ T cells in their lungs at 72 h p.c.

The number of CD4+ αβ T cells is highly upregulated in vaccinated Mut/WT mice. (A) In situ IF staining was performed with frozen lung sections from mock-infected control, nonvaccinated PBS/WT, or vaccinated Mut/WT mice at various...

The number of CD8+ αβ T cells is upregulated in vaccinated Mut/WT mice. (A) In situ IF staining was performed with frozen lung sections from mock-infected control, nonvaccinated PBS/WT, or vaccinated Mut/WT mice at various times...

Absence of hypercytokinemia after WT challenge in vaccinated C57BL/6 mice, while the initial delay remained intact.

In order to better understand the host mediators that may contribute to the survival of vaccinated mice, a multiplex assay that was designed for quantitative measurement of several cytokines, chemokines, and metabolites at the protein level in tissue samples was utilized. Similar to our previous findings (40), the initial inability of the PBS/WT mice to elicit changes in the production of the typical inflammatory mediators was followed by overwhelming increases in the amounts of these mediators at later stages of infection (Fig. ​(Fig.9;9; see Table S1 in the supplemental material). The levels of almost all of the biomarkers tested were upregulated severalfold at 72 h p.c. in the lungs of these mice. The simultaneous increases in the levels of these mediators in conjunction with detection of bacteria in the blood at 72 h p.c. indicated that there was a severe sepsis-like condition, as described previously (40, 54). Intriguingly, vaccination with the mutant did not have any effect on the initial delay in the generation of immune responses in WT challenged mice as no cytokine response was observed in Mut/WT mice at 6 h and 24 h p.c. (Fig. ​(Fig.9).9). At 72 h p.c., in contrast to the PBS/WT mice, the vaccinated Mut/WT mice displayed a more regulated discriminating immune response with differential expression of several host mediators. Notably, the amounts of IFN-γ, interleukin-17 (IL-17), IL-1α, IL-1β, lymphotactin, macrophage-derived chemokine, and RANTES (CCL5) were 4-fold (P = 0.008), 4-fold (P = 0.005), 3-fold (P = 0.001), 2-fold (P = 0.001), 2.5-fold (P = 0.05), 2.4-fold (P = 0.05), and 2-fold (P = 0.05) greater, respectively, than the amounts in the PBS/WT mice. On the other hand, the amounts of granulocyte-macrophage colony-stimulating factor, IL-6, IL-18, KC-Gro-α (CXCL1), monocyte chemoattractant protein 1 (MCP-1) (CCL2), MCP-3 (CCL7), and MIP-2 (CXCL2) were 9-fold (P = 2.0e-3), 7-fold (P = 1.0e-3), 6-fold (P = 4.0e-4), 9-fold (P = 7.0e-4), 7.7-fold (P = 1.0e-3), 6.7 (P = 1.0e-3) fold, and 9-fold (P = 8.0e-5) less, respectively, in Mut/WT mice than in PBS/WT mice. Although the levels of IL-10 and TNF-α were higher in lungs of PBS/WT mice than in Mut/WT mouse lungs at 72 h p.c., the differences were not statistically significant. However, the levels of IL-10 in Mut/WT mouse lungs dropped at 120 h p.c. and were significantly lower (P = 0.005) than those in PBS/WT mouse lungs at that time point. These results indicate that while lethal infection with the WT bacteria resulted in hypercytokinemia reminiscent of sepsis, vaccination with the Mut bacteria elicited regulated upregulation of mainly the Th1 type of cytokines, which are known to protect against intracellular bacterial infections.

Intranasal vaccination with the mutant induces a predominant Th1 type of antibody response after challenge with WT bacteria.

Sera were collected from Mut/WT mice at 2 weeks p.c., and the titers of IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA were determined by performing ELISA. To determine antibody isotype responses specifically elicited by challenge with WT bacteria, sera from mice vaccinated with Mut for 3 weeks and challenged with the same dose of Mut (Mut/Mut mice) were collected at 2 weeks p.c. for comparison. Analysis of antibody responses in mice infected with the mutant alone at 3 weeks p.i. (Mut-3wk mice) showed that the levels of IgM, IgG2a, IgG2b, and IgG3 were greater than those in PBS-inoculated control animals (Fig. ​(Fig.10).10). These levels reflect the baseline levels of the antibody isotypes analyzed at the time of challenge (Fig. ​(Fig.10).10). Comparison of these mice with the Mut/Mut mice showed that the challenge with Mut elicited increased levels of IgG1, IgG2a, and IgG3, although the differences were not statistically significant. In contrast, intranasal challenge of vaccinated mice with the WT bacteria specifically elicited a more robust IgG2a and IgG2b response indicative of the Th1 antibody response (Fig. ​(Fig.10).10). The levels of these isotypes were significantly higher in these mice than in Mut-3wk or Mut/Mut mice. The levels of IgG3 and IgA were also somewhat increased in Mut/WT mice after challenge, but the differences were not statistically significant. These results further highlight the finding that there was induction of a Th1 type of response after intranasal vaccination with the Mut bacteria in the form of serum antibody presumably involved in protection.

Vaccination with Mut induces a predominant Th1-type antibody response. Mice were intranasally vaccinated with 3 × 102 CFU of Mut for 3 weeks, and this was followed by challenge with similar doses of WT bacteria (Mut/WT) or Mut bacteria (Mut/Mut)....

Kinetics of upregulation of S100A9, a DAMP protein, are altered in Mut/WT mice.

The levels of a recently described danger-associated molecular pattern (DAMP) protein associated with severe sepsis in the lungs of PBS/WT mice were compared with those in the lungs of Mut/WT mice by in situ IF staining, as well as by Western blot analysis. Very few cells stained positive for S100A9 in mock-infected control lungs (Fig. 11A1 and A1′). Following a baseline expression level similar to that in mock-infected control mice at 6 h and 24 h p.c. in PBS/WT mice (Fig. 11A2 and A3), the protein level of S100A9 increased substantially at 72 h p.c. (Fig. 11A4), similar to our earlier observation (40, 54). In lungs of these mice the S100A9 expression seemed to be associated with the lesion areas, as well as lung parenchyma, at 72 h p.c. The levels of this host mediator remained relatively unchanged in vaccinated mice after WT challenge at 6 h and 24 h p.c. (Fig. 11A2′ and A3′). At 72 h p.c., the levels of S100A9 were upregulated mainly in the areas of infiltration in Mut/WT mice (Fig. 11A4′). However, most of this mediator was localized in the cytosol of infiltrating cells that appeared to be viable, in contrast to the results for PBS/WT mice, in which most of S100A9 staining was extracellular and not associated with live cells.

Expression of S100A9, a DAMP protein, in the lungs of mock-infected, Mut/WT, and PBS/WT mice. (A1 and A1′) S100A9 expression in mock-infected control lungs. (A2, A3, and A4) PBS/WT lungs at 6, 24, and 72 h p.c., respectively. (A2′, A3′,...

Analysis of S100A9 in lung homogenates from Mut/WT and PBS/WT mice showed that there were similar kinetics of upregulation until 72 h p.c. However, at 120 h p.c. the levels of the S100A9 protein were lower in Mut/WT mice than in PBS/WT mice (Fig. 11B), which is consistent with resolution of the infection.

DISCUSSION

F. tularensis subsp. tularensis is considered a potential bioweapon owing to its extreme virulence, its low infectious dose, and the ease of its transmission (43). The virulence mechanism(s) resulting in mortality associated with this organism and the correlates of protective immunity are not well understood. Understanding the host determinants of disease progression is essential for designing effective therapeutics for use against this highly infectious intracellular bacterium. The murine model organism F. novicida induces a disease in mice which mimics human type A tularemia and thus has served as an important model for understanding the host-pathogen interactions (25, 39, 40). In the current study, the host responses after intranasal immunization with an attenuated and protective transposon mutant of F. novicida strain U112 lacking a 58-kDa hypothetical protein were compared with the host responses after lethal challenge with the wild-type strain via the same route. Our results show that (i) the presence of severe sepsis associated with sustained bacteremia, hypercytokinemia, and widespread leukocyte apoptosis and (ii) depletion of T lymphocytes are associated with the lethal outcome of respiratory tularemia, whereas an absence of these correlates is associated with survival. In addition, a robust cell-mediated and humoral Th1 type of response was observed in mice protected from respiratory tularemia.

The 58-kDa hypothetical protein belongs to a family of five hypothetical proteins of Francisella that are unique to this organism (38). A defined genetic mutant of type A strain SchuS4 of F. tularensis subsp. tularensis lacking the gene encoding this 58-kDa hypothetical protein and a spontaneous type A mutant harboring a defective version of this gene have been shown to be attenuated for infection in mice (63). Intradermal immunization with the deletion mutant protected mice against aerosol challenge with fully virulent strain SchuS4. However, the host immune responses associated with this protection were not characterized. In the current study, the intranasal inoculation route was chosen for immunization with the mutant lacking the 58-kDa protein gene since respiratory tularemia is the most lethal form of the disease, which makes it important to understand the disease in the context of the lung microenvironment. Our previous studies showed that this mutant is attenuated for growth in murine macrophages, replicates in vivo, and causes a subclinical infection in vivo (54).

In the present study, intranasal immunization of mice with the mutant lacking the 58-kDa protein gene provided protection against a subsequent intranasal challenge with a lethal dose of WT bacteria. Although the vaccinated mice exhibited systemic dissemination to the liver and spleen, the bacterial replication was less than that in the unvaccinated mice, and the infection was cleared by 120 h p.c. The control of bacterial replication was reflected by reduced tissue damage and much less pathology in these mice. Extensive tissue pathology is a major complication of acute respiratory infections that are associated with severe sepsis (45, 46). Unbridled production of inflammatory immune mediators during severe sepsis can result in capillary leakage, tissue injury, lethal organ failure, and mitochondrial dysfunction leading to inhibition of oxidative respiration (6, 12, 18, 51, 62). The overt loss of immune cells by extensive cell death compromises the host's ability to eradicate the infectious agent, leading to unchecked replication of the pathogen (31, 50). During respiratory F. novicida infection in mice, the rapid rate of bacterial replication is followed by systemic dissemination and bacteremia (40, 54). Concomitant hypercytokinemia, a condition most commonly associated with severe sepsis characterized by excessive production of host mediators, is observed in these mice. Our recent studies show that respiratory infection with the type A human pathogen F. tularensis strain SchuS4 also results in severe sepsis marked by systemic dissemination, bacteremia, and hypercytokinemia (data not shown). In the current study, the protected Mut/WT mice did not exhibit bacteremia at any time after challenge tested. In addition, hypercytokinemia was not present in the lungs of protected Mut/WT mice. These mice exhibited differential upregulation of cytokines like IFN-γ, IL-1α, and IL-1β. IFN-γ and IL-1β are known to protect against Francisella infection (5, 11, 35, 39, 41); however, the role of IL-1α in Francisella infection is not clear. Since IL-1α and IL-1β bind to the same receptor, it is generally assumed that the two forms of the cytokine elicit similar responses. However, it has been shown that IL-1α plays a distinct role in intestinal inflammation caused by Yersinia enterocolitica (13). In the current study IL-1α was upregulated more than IL-1β in both vaccinated and nonvaccinated mice. Also, vaccination with Mut resulted in a greater increase in the level of IL-1α (threefold) than in the level of IL-1β (twofold) after challenge with the WT bacteria. Possibly the two forms of IL-1 play different roles during Francisella infection. Nonetheless, both cytokines seem to be associated with the protective response during respiratory tularemia. Importantly, the PBS/WT mice exhibited unbridled upregulation of multiple cytokines, chemokines, and other immune mediators, including IL-6, MCP-1 (CCL2), and KC-Gro-α (CXCL1), which are classical markers of severe sepsis (27). This indicated that protection from complications of severe sepsis is central to survival during respiratory tularemia.

In the current study, a robust increase in the level of the S100A9 protein was observed during WT infection in the lungs of mice. Immune recognition of damaged tissue during an inflammatory response is mediated by intracellular mediators released from dying cells, which are known as DAMP proteins. The extracellular location of these “alarmins,” which are sequestered within the cells under normal conditions, leads to overwhelming stimulation of the immune system via pattern recognition receptors, such as Toll-like receptors. This converts an otherwise beneficial response into excessive damaging inflammation (19, 49). The S100A9 protein, which is one such DAMP protein, is reported to induce neutrophil chemotaxis (52), mediate apoptosis (23), and activate the NFκB pathway (60). In the present study, IF staining showed that extracellular localization of this alarmin in lesion areas coincided with extensive cell death observed by TUNEL staining in WT-infected lungs. The interaction of this and possibly other DAMP proteins (40) with pattern recognition receptors likely amplifies the inflammation and other tissue damage in PBS/WT mice. However, S100A9, along with other members of the S100 family of immune mediators, also has protective roles, such as antimicrobial activity (58), regulation of matrix metalloproteinase activity (34), and wound repair (61, 67). The protected Mut/WT mice displayed increased levels of S100A9 at 72 h p.c., which was largely localized in the infiltrating cells. The absence of this alarmin extracellularly is consistent with the reduced level of cell death observed in these mice and perhaps reflects its more reparative role rather than its inflammatory role.

Massive depletion of αβ T cells from lungs of PBS/WT mice was observed at 72 h p.c. compared to mock-infected control mice. In contrast, Mut/WT mice contained increased numbers of these cells compared to mock-infected control or PBS/WT mice at this time point. Th1 responses are important for controlling primary infection with Francisella, as well as for long-term immunity against Francisella infection (16). IFN-γ secreted by Th1 cells activates myeloid cells and increases their ability to clear intracellular bacterial pathogens (21). In this study, although the cellular source of Th1 cytokines in Mut/WT mice was not determined, the increased levels of IFN-γ in these mice were likely due to αβ T cells since the number of these cells increased substantially after challenge in these mice. Moreover, upregulation of the IgG2a and IgG2b levels in sera of vaccinated mice indicated that there was a Th1 cytokine-dependent antibody response in protected mice. Although the exact role of these antibody isotypes elicited by vaccination with Mut remains to be determined, the opsonizing ability of Francisella-specific antibodies has been shown to facilitate clearance of the pathogen in strain LVS-vaccinated humans, as well as in experimental animals infected with this and other strains of Francisella (for a review, see reference 16).

Depletion of αβ T cells occurred in lungs of nonvaccinated PBS/WT mice, despite massive infiltration of other immune cells, such as CD11b+ and Gr1+ cells. Our previous studies have shown that depletion of αβ T cells, most notably CD4+ T cells, during lethal respiratory infection with F. novicida is due to apoptosis (55). An increased level of apoptosis in lymphocytes has also been observed during experimental sepsis, as well as in patients (28, 30). Moreover, inhibition of lymphocyte apoptosis improves survival during sepsis (37, 47). This depletion of lymphocytes contributes to late-stage “immunoparalysis” observed during sepsis, which shuts down the adaptive immune responses required to fight the infection (29, 32, 33). Also, a recent study indicated that T-cell-mediated suppression is required for tempering the strength of innate immune responses to limit damage to the host (36). In our studies the PBS/WT mice exhibited depletion of CD4+ and CD8+ T cells in lungs with concomitant bacteremia, suggesting that T cells play an early key role in limiting bacteremia. This notion is supported by a recent study in which depletion of CD4+ and CD8+ T cells in human sepsis patients was observed, and in the same study mice deficient in CD4+ T cells had increased mortality, increased bacterial loads, and decreased neutrophil activity resulting from sepsis (42). Thus, during lethal Francisella infection, the loss of T cells likely contributes to the host's inability to control the infection, either due to a lack of T lymphocytes tempering the strength of the innate immune response or due to loss of the normal transition from an innate response to an adaptive response. Both CD4+ and CD8+ T cells have been shown to control Francisella infection in an IFN-γ- and TNF-α-dependent manner (1, 9, 66). In our studies the number of CD4+ αβ T cells was higher than the number of CD8+ αβ T cells in both vaccinated and nonvaccinated mice. Since macrophages are the primary cell types infected by Francisella, a higher number of CD4+ T cells indicates that there are major histocompatibility class II-dependent mechanisms of bacterial killing, possibly mediated by reactive oxygen and nitrogen species. Other cell types, such as dendritic cells, support Francisella infection but are not good producers of reactive oxygen and nitrogen species. Instead, they activate CD8+ T-cell-mediated cytolytic responses via major histocompatibility class I. Nevertheless, the increased numbers of αβ T cells in vaccinated Mut/WT mice may be the result of increased infiltration of these cells in lungs and/or increased localized proliferation of T cells in response to improved antigen presentation since Francisella infection is known to interfere with the maturation of antigen-presenting cells (3). These observations indicate that the absence of severe sepsis and robust proliferation and infiltration of T cells in protected Mut/WT mice are beneficial for survival after an otherwise lethal challenge.

In this study, the PBS/WT and Mut/WT mice displayed similar initial delays in the upregulation of immune mediators. Although the initial delay in activation of host immune responses during respiratory Francisella infections has been postulated to be a virulence mechanism (2, 39, 40), this phenomenon does not seem to be sufficient for the lethal outcome of the disease. Thus, the PBS/WT mice succumbed to infection, but Mut/WT mice did not develop overt disease (survival rate, 100%) despite a similar initial delay. The delay in the initial inflammatory immune responses compared with the responses to other intracellular organisms may be involved in virulence. However, our results strongly suggest that T-cell depletion and a subsequent ineffective innate-to-adaptive transition lead to bacteremia and sepsis and ultimately to death.

Taken together, our studies indicate that a severe sepsis-like condition elicited by bacteremia, hypercytokinemia, and T-cell depletion occurring at the later stages of infection is likely responsible for the mortality associated with Francisella infection. In contrast, the absence of such correlates coupled with a robust Th1 response is protective. This study provides insights important for designing vaccine strategies and therapeutic interventions for this disease.